Category Archives: C. diff. Research & Development

It’s surprisingly common for people to wrongly think they have a penicillin allergy — and that misconception can be dangerous for their health.

Ten percent of all patients in the United States claim to have a penicillin allergy. Of those people, 90 percent are not truly allergic and can tolerate the drug. That means millions of people take alternative antibiotics, which are more expensive and can put their health and potentially the health of others at risk. The solution is a simple allergy test.

A study in the British Medical Journal (BMJ) looked at six years’ worth of medical records for patients in the United Kingdom and found that those with a penicillin allergy had an almost 70 percent greater chance of acquiring a methicillin-resistant Staphylococcus aureus (MRSA) infection and a 26 percent increased risk of Clostridium difficile-related colitis (C. diff.). MRSA and C. diff. are major health risks worldwide. The study compared adults with a known penicillin allergy to similar people without a known penicillin allergy.

People labeled with a penicillin allergy are usually instead given broad-spectrum antibiotics, which may kill off more good bacteria along with the bad. This appeared to increase a patient’s risk of infection with MRSA or C. diff., whichare common in our environment and can live without causing any problems on someone’s skin or gut. However, if a broad antibiotic kills off competing good bacteria, MRSA and C. diff. can thrive and start to cause problems.

“Penicillin-related drugs, that whole class … they’re very effective at killing, and they’re very targeted. So for some bacteria they’re still the best. Oldie but goody,” said Kim Blumenthal, lead author of the new study and assistant professor of medicine at Harvard Medical School.

“I have seen so many terrible, terrible outcomes” from C. diff. infections, Blumenthal said, including serious diarrhea, sepsis and death.

“All of us need to understand that antibiotic use is not a free ride, it carries a lot of risk,” said Paul Sax, clinical director of infectious diseases at Brigham and Women’s Hospital. He was not involved in the study but he says the study adds to the “substantial body of evidence” which shows that a penicillin allergy has been linked to longer hospital stays and an increased risk of acquiring resistant infections.

Using non-targeted antibiotics can quickly breed resistant bacteria. “Not only is it harmful to the world and the general population . . . but it’s harmful to the individual patient. So the message to the public is that it could be dangerous to you or me,” said Helen Boucher, director of the Infectious Diseases Fellowship Program at Tufts Medical Center, who was not involved in the study.

“In antibiotic resistance we don’t have a very loud patient advocacy voice . . . and the reasons for that are complicated, but a lot of it has to do with the fact that a lot of the victims aren’t here to speak for themselves because they died,” Boucher said.

The infections are resistant to many known drugs and can quickly become life-threatening. According to the Centers for Disease Control and Prevention, 2 million people, equivalent to the approximate population of Brooklyn, are infected with resistant bacteria every year. At least 23,000 people die each year as a direct result and even more from complications.

Diagnosing penicillin allergies is challenging. Symptoms such as a rash, nausea or diarrhea could be a sign of allergy, or they might coincidentally occur when someone is taking antibiotics, according to Jonathan Grein, medical director at the Department of Hospital Epidemiology at Cedars Sinai Medical Center in Los Angeles. Children frequently get rashes that are mistaken for penicillin allergies, Blumenthal said.

Even if people are diagnosed correctly as children, they can grow out of an allergy, said Sax.

Which raises the question, what exactly is an allergy? The Internet is full of “answers,” as any late-night Googling hypochondriac can tell you, but an allergy is simply an exaggerated immune response triggered unnecessarily. It can be anything from a rash to trouble breathing.

“Part of the problem is that ‘allergy’ means different things to different people,” said Grein. “Making that distinction between these intolerances and side effects and life-threatening immediate allergic reaction, that’s where the challenge is.”

For example, a patient of Sax’s, in his mid-20s, had a life-threatening heart infection. Penicillin could save him, but his medical record said he was allergic to the drug. Careful questioning by his medical team was able to determine that although he had nausea and diarrhea while on penicillin, he did not have an allergy, Sax said. Knowing this, the hospital administered the appropriate penicillin antibiotic to save his life.

In the case of penicillin, it is important to know that the risks of the allergy diagnosis are sometimes worse than the symptoms of the allergy itself. In most cases, penicillin should only be avoided if the allergy is immediate and life-threatening.

“There are over 30 million Americans who have a penicillin allergy on their record. And there are things we can do,” Blumenthal said.

Examine your own medical record, Blumenthal said. “I would want patients to think, ‘Hmm, am I really allergic to penicillin, or did my mom just tell me and it’s not really true, and should I get that evaluated?’ ”

If it’s been more than 10 years since you were diagnosed, talk to your doctor about getting retested.

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Though recurrent Clostridium difficile infections (CDI) are common and pose a major clinical concern, data are lacking regarding mortality among patients who survive their initial CDI and have subsequent recurrences. Risk factors for mortality in patients with recurrent CDI are largely unknown.

Methods

Veterans Affairs patients with a first CDI (positive C. difficile toxin(s) stool sample and ≥ 2 days CDI treatment) were included (2010–2014). Subsequent recurrences were defined as additional CDI episodes ≥ 14 days after the stool test date and within 30 days of end of treatment. A matched (1:4) case-control analysis was conducted using multivariable conditional logistic regression to identify predictors of all-cause mortality within 30 days of the first recurrence.

In our national cohort of Veterans, crude mortality decreased by 44% from the initial episode to the third recurrence. Treatment with antibiotics, PPIs, and underlying co-morbidities were important predictors of mortality in recurrent CDI. Our study assists healthcare providers in identifying patients at high risk of death after CDI recurrence.

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Objectives

This study aimed to examine changes to the microbiota composition and metabolic profiles of seven patients with recurrent Clostridium difficile infection (rCDI), following treatment with faecal microbiota transplant (FMT).

Summary

Objectives

This study aimed to examine changes to the microbiota composition and metabolic profiles of seven patients with recurrent Clostridium difficile infection (rCDI), following treatment with faecal microbiota transplant (FMT).

Methods

16S rDNA sequencing and 1H NMR were performed on faecal samples from the patients (pre-, post-FMT, and follow-up) and the associated donor samples. Sparse partial-least-square analysis was used to identify correlations between the two datasets.

Results

The patients’ microbiota post-FMT tended to shift towards the donor microbiota, specifically through proportional increases of Bacteroides, Blautia, and Ruminococcus, and proportional decreases of Enterococcus, Escherichia, and Klebsiella. However, although cured of infection, one patient, who suffers from chronic alcohol abuse, retained the compositional characteristics of the pre-FMT microbiota. Following FMT, increased levels of short-chain fatty acids, particularly butyrate and acetate, were observed in all patients. Sparse partial-least-square analysis confirmed a positive correlation between butyrate and Bacteroides, Blautia, and Ruminococcus, with a negative correlation between butyrate and Klebsiella and Enterococcus.

Conclusions

Clear differences were observed in the microbiota composition and metabolic profiles between donors and rCDI patients, which were largely resolved in patients following FMT. Increased levels of butyrate appear to be a factor associated with resolution of rCDI.

Introduction

Although Clostridium difficile is present in the intestines of ∼3–5% of healthy adults,1 the occurrence of C. difficile infection (CDI) in healthy individuals is relatively uncommon due to the protective effect of the gut microbiota. The incidents and severity of CDI has risen significantly over the last decade, and it is now recognised as the main causative agent of healthcare-associated infectious diarrhoea in hospitals worldwide.2 The standard treatment for CDI is the administration of metronidazole for mild to moderate infections, and oral vancomycin or fidaxomicin for severe infections and relapses. The ability of C. difficile to form spores, coupled with the increase in antibiotic-resistant strains, can lead to persistence of infection, relapses, and the administration of more antibiotics, which further depletes the commensal bacteria. This creates an environment that is more favourable to C. difficile, thus setting up a cycle of relapse and re-infection. It is estimated that 20-30% of patients who develop a first episode of CDI go on to have at least one relapse, and of these, a further 60% develop further episodes of relapses.3 This increases the need for further antibiotics, the risk of antibiotic-resistance in the gut commensal flora, and costs to the health service, with each episode of CDI estimated to cost approximately £7000 in 2010.4

Faecal microbiota transplants (FMT) represents an effective alternative to antibiotics to treat recurrent CDI (rCDI), with a primary cure rate as high as 91%.5 The central tenet behind FMT is that the introduction of a healthy bacterial community into the intestines produces an environment that is less favourable to C. difficile by increasing colonisation resistance and reinstating a protective effect. The advantages of this treatment are that it is quick, cost-effective, and could help to eradicate antibiotic resistant strains of C. difficile.

It is known that a dysbiotic gut microbiota increases the risk of developing CDI, however whether there is a common element within this community composition that could help to determine if a patient is at greater risk of rCDI is as yet unknown. The reduction in diversity within the dysbiotic gut microbiota would also suggest a reduction in metabolic potential through the loss of gene diversity. The functional redundancy6 within the gut microbiota suggests, that metabolic function is more relevant than which species are present or absent. Whilst a number of studies have looked at the changes in microbiota composition due to FMT,7,8,9,10 we know little about the changes to the metabolic capacities of the altered microbiota. The aim of this study was to assess FMT-induced changes in both the microbial community structure and metabolite profiles of the gut microbiomes of seven patients with rCDI, as well as those of their associated FMT donors.

Patients and methods

Patients

Patients were selected as candidates for the FMT procedure if they had at least two confirmed recurrences of CDI. C. difficile testing was based on a two stage algorithm in line with Public Health England recommendations.11 This involves screening faecal samples by glutamate dehydrogenase enzyme immunoassay (Techlab, USA), followed by C. difficile toxin testing by enzyme immunoassay (Techlab, USA). Glutamate dehydrogenase positive, toxin negative samples were further tested for the presence of toxigenic genes by PCR. FMT exclusion criteria included immunocompromised patients, those aged less than 16, and those with severe comorbidities which would make the patient unfit for endoscopy. FMT was introduced into clinical care at Norfolk and Norwich University Hospital following approval by the New Therapies committee, and was performed in accordance with the Helsinki Declaration of 1975. Patients were consented for the study by a clinician following a detailed discussion of the procedure with the patient or their next of kin. All patient data is fully anonymised.

Donor screening

The faecal donors used for the cohort of patients who underwent FMT in this study were both healthy Caucasian males with a BMI between 24 and 27 kg/m2, aged 36 (D05) and 30 (D03) years of age, respectively. Potential donors were asked to complete a questionnaire adapted from van Nood et al.12 regarding their medical history and lifestyle habits to identify risk factors for potentially transmittable diseases. Eligible candidates provided blood and stool samples for laboratory screening tests. Blood samples were screened for hepatitis A, B, C, and E antibodies, HIV 1 & 2, human T-lymphotropic virus 1 & 2, Epstein-Barr virus, Cytomegalovirus, syphilis, Entamoeba histolytica, Strongyloides stercoralis, and Treponema pallidum. Stool samples were tested for the presence of C. difficile or its toxins, Helicobacter pylori antigen, Norovirus, methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, extended-spectrum β-lactamase-producing organisms, carbapenemase-producing Enterobacteriaceae, Escherichia coli O157, Salmonella spp., Shigella spp., and Campylobacter species. In addition, microscopy was used to investigate for ova, cysts, and parasites. Prior to the donation of stool samples for each FMT procedure, donors were asked to refrain from eating peanuts and shellfish, and to complete a short screening questionnaire to confirm no changes to health or lifestyle since the last donor screening that may put the patient at risk.

Faecal suspension preparation

Donor faeces were collected in a sterile container on the day of the procedure, and transferred to a sterilised class II safety cabinet (Walker Ltd, UK). A maximum of 80 g of donor stool was homogenised with sterile saline (0.9%), to a ratio of 5 ml saline per gram of stool, in a strainer bag (BA6141/STR; Seward Limited, UK) using a Stomacher® 400 Circulator (Seward Limited, UK) set to 230 RPM for a duration of 1 min. The filtered faecal preparation was drawn up into labelled sterile 60 ml syringes using nasojejunal tubing connected to the Luer lock. The syringes were secured with sterile Luer lock caps and transported immediately to the hospital. Aliquots of the donor faecal sample were immediately stored at −20 °C until analysis.

Faecal suspension infusion

Patients were prescribed oral vancomycin 500 mg four times daily for 4 days, with the last dose received the night before the procedure. Also, on the day before the FMT procedure, a bowel lavage is performed using 4 l of macrogol solution (Klean-Prep, Norgine). Patients were taken to the endoscopy unit for insertion of nasojejunal tube the night before the procedure. Our FMT protocol was adapted from that of van Nood et al.12 On the day of FMT infusion, the patient’s headrest was elevated to 45°, patency of the nasojejunal tube was checked by flushing with water, and 420 ml of faecal suspension was delivered slowly by the patient’s bedside in the isolation room via a nasojejunal tube using the prefilled syringes. This was performed at a rate of ∼20 ml per minute with a break of 5-10 minutes applied halfway through the procedure. Post-infusion instructions were to monitor observations, and record bowel motions. Patients could take on fluids one hour after the procedure, and were observed overnight before discharge the next day at the earliest. Although there are no agreed durations of follow-up post-FMT,13 van Nood et al.12 used two endpoints to measure cure, namely no relapse after 5 weeks, and no relapse after 10 weeks. Resolution was defined as type 4 or less on the Bristol stool chart or stool normal for the patient e.g. in case of percutaneous endoscopic gastrostomy feeding. We followed patients up by telephone or in person if they were re-admitted into the hospital for an unrelated illness. Post-FMT samples were collected after a minimum of 10 days post-FMT, and postal kits were provided to patients who were willing to donate a ‘follow-up’ sample up to 2 weeks later.

Sample analysis

Faecal microbiota analysis

Faecal samples were collected from recipients within 9 days prior to FMT, however the pre-FMT sample for patient R13 was not collected within this timeframe, and a previously frozen sample obtained whilst the patient was suffering from the same episode of CDI was used. Further samples were collected for all recipients following the procedure (‘post-FMT’ range: 11–141 days; ‘follow-up’ range: 4–14 days after post-FMT sample), and stored at −20 °C until analysis. The DNA was extracted using the FastDNA SPIN Kit for Soil (MP Biomedicals, UK) with a bead-beating step.14 DNA yield was quantified using the Qubit fluorometer prior to the samples being sent to the Earlham Institute (UK), where the V4 hypervariable region of the 16S rRNA genes were amplified using the 515F and 806R primers with built-in degeneracy.15 The amplicons were sequenced using paired-end Illumina sequencing (2 × 250 bp) on the MiSeq platform (Illumina, USA). Sequencing data, for the 21 samples that had an appropriate level of sequencing depth, were analysed using the Quantitative Insights Into Microbial Ecology (QIIME) 1.9 software and RDP classifier 16S rRNA gene sequence database.16,17 The trimmed reads were filtered for chimeric sequences using ChimeraSlayer, bacterial taxonomy assignment with a confidence value threshold of 50% was performed with the RDP classifier (version 2.10), and trimmed reads clustered into operational taxonomic units at 97% identity level. Alpha diversity and rarefaction plots were computed using the Chao1 index. Weighted and unweighted UniFrac distances were used to generate beta diversity principal coordinates analysis plots, which were visualised using the Emperor tool.

Faecal metabolite analysis

A known mass (∼ 100 mg) of thawed faecal samples were added to sterile tubes. The faecal waters were generated by adding the phosphate buffer (prepared in D2O) to 8.3% w/v. Homogenised faecal waters were centrifuged at 16,200 x g at room temperature for 5 min. The supernatants were filter sterilised (0.2 µm) and placed in a 5 mm NMR tube. The 1H NMR spectra were recorded at 600 MHz on a Bruker Avance spectrometer (Bruker BioSpin GmbH, Germany) running Topspin 2.0 software and fitted with a cryoprobe and a 60-slot autosampler. Each 1H NMR spectrum was acquired with 1280 scans, a spectral width of 12,300 Hz, and an acquisition time of 2.67 s. The “noesypr1d” pre-saturation sequence was used to suppress the residual water signal with a low-power selective irradiation at the water frequency during the recycle delay and a mixing time of 10 ms. Spectra were transformed with a 0.3 Hz line broadening, and were manually phased, baseline corrected, and referenced by setting the TSP methyl signal to 0 ppm. The metabolites were quantified using the software Chenomx® NMR Suite 7.0TM.

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Main outcome measures The primary outcome was risk of incident MRSA and C difficile. Secondary outcomes were use of β lactam antibiotics and β lactam alternative antibiotics.

Results: Among 64 141 adults with penicillin allergy and 237 258 matched comparators, 1365 developed MRSA (442 participants with penicillin allergy and 923 comparators) and 1688 developed C difficile (442 participants with penicillin allergy and 1246 comparators) during a mean 6.0 years of follow-up.

Among patients with penicillin allergy the adjusted hazard ratio for MRSA was 1.69 (95% confidence interval 1.51 to 1.90) and for C difficile was 1.26 (1.12 to 1.40). The adjusted incidence rate ratios for antibiotic use among patients with penicillin allergy were 4.15 (95% confidence interval 4.12 to 4.17) for macrolides, 3.89 (3.66 to 4.12) for clindamycin, and 2.10 (2.08 to 2.13) for fluoroquinolones. Increased use of β lactam alternative antibiotics accounted for 55% of the increased risk of MRSA and 35% of the increased risk of C difficile.

Conclusions Documented penicillin allergy was associated with an increased risk of MRSA and C difficile that was mediated by the increased use of β lactam alternative antibiotics. Systematically addressing penicillin allergies may be an important public health strategy to reduce the incidence of MRSA and C difficile among patients with a penicillin allergy label.

Introduction

One third of patients report a drug allergy (ie, adverse or allergic reaction),1 the most commonly implicated drug being penicillin and documented in 5-16% of patients.12345 Being labelled with a penicillin allergy affects future prescribing for infections in both outpatients and inpatients, with prescribed antibiotics often more broad spectrum and toxic.2678 Unnecessary use of broad spectrum antibiotics leads to the development of drug resistant bacteria, including meticillin resistant Staphylococcus aureus (MRSA), and healthcare associated infections such as Clostridium difficile related colitis.910111213

Most patients with a documented penicillin allergy are not allergic—that is, there is no immediate hypersensitivity.1415 After evaluation by an allergist, about 95% of patients with reported penicillin allergies were found to be penicillin tolerant.14 The discrepancy between labelled and confirmed penicillin allergy stems from misdiagnosis (eg, a viral exanthem is misinterpreted as an allergy), misassumptions (eg, an intolerance, such as a headache, is listed as an allergy), and remote timing of the allergy evaluation, since 80% of patients with immediate hypersensitivity to penicillin are no longer allergic after 10 years.16 Most patients with a penicillin allergy label therefore unnecessarily avoid penicillins, and often other related β lactam antibiotics, such as cephalosporins.67

To evaluate the public health consequences of having a penicillin allergy label, we conducted a population based matched cohort study and examined the relation between a newly recorded penicillin allergy and the risk of incident MRSA and C difficile.

Methods

Data source

We used data from the Health Improvement Network (THIN), an electronic medical record database of 11.1 million patients registered with general practices in the United Kingdom. Because the National Health Service requires people to register with a general practice regardless of health status, THIN is a population based cohort representative of the UK general population.17 During consultations with patients in primary care, general practitioners (GPs) enter clinical data, including height, weight, smoking status, diagnoses, and prescription drugs. Patient diagnoses are recorded using READ codes, the UK’s standard clinical terminology system.18 Drug allergies are linked to a drug prescription, or recorded as a diagnosis (eg, personal history of penicillin allergy). The GP enters details of the drug allergy, including reaction type, severity of reaction, and certainty of diagnosis. All GPs are trained in data entry, with the quality of their data periodically reviewed. Previous studies using THIN have confirmed the validity of both prescriptions and diagnoses.1920

Study design

We performed a matched cohort study among participants aged more than 18 years, who were enrolled in the THIN database between 1995 and 2015. Eligible participants had no history of MRSA or C difficile diagnoses before study entry and were required to have at least one year of enrolment with a general practice before entering the study to allow for assessment of exposure and covariates. We identified adults with their first recorded penicillin allergy and selected up to five penicillin users without a penicillin allergy matched on age (one year either way), sex, and study entry time (within one year either way). Such comparators were chosen to further ensure the comparability of indications for penicillin use (eg, infection tendency) and associated features. The index date for cases was the date of first entry of an allergy diagnosis in the THIN database; the matched index date for comparators was within one year of a penicillin prescription.

Assessment of exposure and outcomes

The exposure of interest was a documented penicillin allergy, defined as an allergy to a penicillin antibiotic linked to a penicillin prescription, or one or more relevant READ diagnosis codes for a penicillin allergy or adverse effect (see supplemental table 1).

The primary outcomes were incident cases of doctor diagnosed MRSA and C difficile during the follow-up period. We identified MRSA and C difficile by the presence of one or more relevant READ diagnosis codes.21222324 For MRSA, codes indicated MRSA infection, carriage, eradication, or decontamination whereas for C difficile, codes indicated C difficile infection or detection of antigen or toxin (see supplementary table 1).

We also assessed antibiotic utilization during the follow-up period, derived from the prescription record. We grouped all antibiotics prescribed into classes: penicillins, first generation cephalosporins, macrolides, clindamycin, fluoroquinolones, tetracyclines, and sulfonamides. Given that vancomycin, aminoglycosides, and linezolid are commonly administered parenterally and therefore seldom administered to outpatients by GPs, we assessed these antibiotics separately.

Assessment of covariates

We identified demographic and lifestyle factors before the index date, such as age, sex, body mass index, socioeconomic status, smoking status, and alcohol use. READ diagnosis codes at the index date were used to ascertain relevant comorbidities (diabetes, renal disease, hemodialysis, malignancy, liver disease, and infection with human immunodeficiency virus (HIV)) and to calculate the adapted Charlson comorbidity index25 at baseline. Using the prescription records, we identified the number of antibiotics prescribed in the year before the index date and whether proton pump inhibitors or systemic corticosteroids were used at baseline. Concomitant allergies to cephalosporin antibiotics and other antibiotics were linked to prescriptions or identified using READ diagnosis codes. We determined participants who were residents of nursing homes at baseline. Finally, we calculated the number of visits to a GP and hospital admissions during the year before the index date.

Statistical analysis

We compared baseline characteristics between participants with penicillin allergy and their comparators. Follow-up time for each participant was calculated from the index date to the date of one of several events: the study endpoints (MRSA or C difficile), death, or end of the study (31 December 2015), whichever occurred first.

We identified incident MRSA cases and number of person years of follow-up for each cohort separately. We calculated the hazard ratios for the relation of penicillin allergy status to the risk of MRSA using Cox proportional hazard models. In the multivariable Cox model we adjusted for age, sex, body mass index, socioeconomic status, smoking status, alcohol use, Charlson comorbidity index, hemodialysis, number of antibiotic prescriptions, proton pump inhibitor use, corticosteroid use, other antibiotic allergies, resident of nursing home, visits to a GP, and admissions to hospital. We repeated the same analyses for the risk of C difficile. We also calculated the absolute risk difference.

In both the penicillin allergy cohort and the comparison cohort we determined the rates of subsequent antibiotic utilization by class. We used Poisson regression models to estimate the incidence rate ratio for the relation of penicillin allergy status to the rates of subsequent antibiotic use, while adjusting for the same covariates.

We performed mediation analyses to examine the extent to which the effect of penicillin allergy status on the risk of MRSA or C difficile was through its effect on utilization of β lactam alternative antibiotics.26 Specifically, we grouped utilization into five categories based on previous studies that evaluated the impact of various antibiotics on the risk of MRSA and C difficile910111213: fluoroquinolones, clindamycin, macrolides, vancomycin, aminoglycosides, and linezolid (all β lactam alternative antibiotics considered in this study); fluoroquinolones, clindamycin, and macrolides; fluoroquinolones and macrolides; fluoroquinolones and clindamycin; and fluoroquinolones alone. Using marginal structural models we then estimated the natural direct effect (ie, the effect of penicillin allergy status on the risk of MRSA or C difficile not through a specific group of antibiotics) and the natural indirect effect (ie, the effect of penicillin allergy status on the risk of MRSA or C difficile through a specific group of antibiotics), while adjusting for the same confounding variables,26 and reported the adjusted risk ratio and percentage mediated.

For all analyses we imputed unknown values for covariates (ie, missing body mass index, alcohol use, and smoking status) using a sequential regression method based on a set of covariates as predictors. To minimize random error, we imputed five datasets and then combined estimates from these datasets by calculating effect estimates from each imputed dataset and then averaging estimates and their confidence intervals using Rubin’s rules.27 All analyses were performed using SAS, version 9.2 (SAS Institute, Cary, NC).

Patient and public involvement

No patients were involved in setting the research question or the outcome measures, nor were they involved in developing plans for implementation of the study. No patients were asked to advise on interpretation or writing up of results. There are no plans to disseminate the results of the research to study participants or the relevant patient community. Individual patient consent was not sought given the use of anonymized data.

Patients with penicillin allergy were similar to their comparators for age, sex, body mass index, socioeconomic status, smoking status, and alcohol use (table 2). They were also similar for diabetes, renal disease, hemodialysis, malignancy, liver disease, HIV, Charlson comorbidity index, previous antibiotic prescriptions, use of proton pump inhibitors and systemic corticosteroids, nursing home residency, visits to a GP, and hospital admissions. Other antibiotic allergies were more common in patients with a penicillin allergy.

Penicillin allergy and risk of MRSA and C difficile

During the mean follow-up time of 6.0 years for patients with penicillin allergy and 6.1 years for comparator patients, 442 patients with penicillin allergy and 923 comparator patients developed MRSA, and 442 patients with penicillin allergy and 1246 comparator patients developed C difficile (table 3 and supplemental table 2).

The age, sex, and study entry time matched hazard ratios for patients with penicillin allergy were 1.84 (95% confidence interval 1.64 to 2.06) for MRSA and 1.37 (1.23 to 1.53) for C difficile. The matched and multivariable adjusted hazard ratios for patients with penicillin allergy were 1.69 (1.51 to 1.90) for MRSA and 1.26 (1.12 to 1.40) for C difficile, respectively. The corresponding adjusted risk differences were 49/100 000 person years for MRSA and 27/100 000 person years for C difficile.

Penicillin allergy and subsequent antibiotic utilization

Patients with penicillin allergy were less often prescribed penicillin than their comparators (adjusted incidence rate ratio 0.30, 95% confidence interval 0.30 to 0.31), but had increased use of macrolide antibiotics (4.15, 4.12 to 4.17), clindamycin (3.89, 3.66 to 4.12]), fluoroquinolones (2.10, 2.08 to 2.13), tetracyclines (1.75, 1.73 to 1.76), and sulfonamide antibiotics (1.26, 1.25 to 1.27; table 4). Though vancomycin, aminoglycosides, and linezolid were overall infrequently prescribed, they were more often prescribed to patients with penicillin allergy than to their comparators (supplemental table 3).

Mediation effects of alternative antibiotic use

Compared with patients who did not receive penicillins, patients receiving penicillins did not have an increased risk of MRSA (adjusted risk ratio 1.07, 95% confidence interval 0.95 to 1.20), but had an increased risk of C difficile (1.18, 1.06 to 1.31; supplemental table 4). Patients receiving macrolide antibiotics had an increased risk of MRSA (1.72, 1.54 to 1.91) and C difficile (1.30, 1.18 to 1.43). Patients receiving clindamycin had an increased risk of MRSA (2.97, 2.11 to 4.16) and C difficile (2.76, 2.00 to 3.81). Patients receiving fluoroquinolones had an increased risk of MRSA (2.38, 2.12 to 2.67) and C difficile (1.72, 1.54 to 1.93).

The effect of a penicillin allergy on the risk of MRSA was 55% mediated through β lactam alternative antibiotic classes; 55% mediated through fluoroquinolones, clindamycin, and macrolides; 54% mediated through fluoroquinolones and macrolides; 26% mediated through fluoroquinolones and clindamycin; and 24% mediated through fluoroquinolones alone (table 5). The effect of penicillin allergy on C difficile was 35% mediated through β lactam alternative antibiotic classes; 26% mediated through fluoroquinolones, clindamycin, and macrolides; 24% mediated through fluoroquinolones and macrolides; 20% mediated through fluoroquinolones and clindamycin; and 16% mediated through fluoroquinolones alone.

Discussion

In this large cohort study reflective of the United Kingdom general population, we found that a penicillin allergy label was associated with a 69% increased risk of MRSA and a 26% increased risk of C difficile. Once documented, a penicillin allergy resulted in increased use of β lactam alternative antibiotics, with a fourfold increased incidence of macrolides and clindamycin utilization, and a twofold increased incidence of fluoroquinolone utilization. Furthermore, more than half of the increased MRSA risk and more than one third of the increased C difficile risk among patients with penicillin allergy was attributable to administered β lactam alternative antibiotics.

Comparison with other studies and policy implications

We found that patients with a penicillin allergy label had nearly a 70% increased risk of new MRSA than their matched comparators, even after adjustment for known MRSA risk factors.28 This provides supporting evidence for a previous US study that showed a 14% increased MRSA prevalence in inpatients who were allergic to penicillin.8 Our result emphasises that outpatient use of antibiotics is strongly associated with the risk of developing MRSA.2228 Consistent with previous studies, we found that β lactam alternative antibiotics increased the risk of MRSA to a greater degree than did penicillins12132930; whereas the mechanism of resistance is not known, the same factors that predispose staphylococcus to develop resistance to meticillin are thought to predispose staphylococcus to multidrug resistance that includes resistance to meticillin.122931 With more than half of the increased MRSA risk among patients with listed penicillin allergy directly attributable to increased outpatient β lactam alternative antibiotic use (largely fluoroquinolones and macrolides), this risk appears modifiable if prescribing patterns among those with penicillin allergy could be altered.

C difficile is responsible for almost one half million infections and 15 000 deaths each year in the US, and the Centers for Disease Control and Prevention consider C difficile one of three urgent threats to public health.32 Patients with penicillin allergy in this study had a 26% increased C difficile risk compared with age, sex, and study entry time matched comparators after adjustment for other known risk factors for C difficile.10333435 This result also corroborates the previous US study, which found a 23% increased C difficile prevalence in hospital patients with a penicillin allergy.8 While other studies similarly identified that clindamycin and fluoroquinolones were associated with the greatest risk of C difficile,91011 we found that 35% of the heightened risk of C difficile in patients with penicillin allergy was directly attributable to use of β lactam alternative antibiotics, with quinolone use alone responsible for 16% of the heightened risk. The mechanism by which antibiotic use precipitates C difficile is through disruption of the host microbiome and creation of an environment where C difficile can overgrow.36 Antibiotics not captured in this dataset (eg, those administered at dialysis or in hospitals) and non-antibiotic risk factors234 are likely responsible for the remainder of C difficile cases. Although current efforts to reduce C difficile largely focus on reducing C difficile infections in hospitals and rehabilitation centers, one third of C difficile infections occur in the community, and occur in outpatients.37 Our findings suggest that more systematic efforts to identify patients with listed penicillin allergy who are not truly allergic to penicillins could help decrease rates of community associated C difficile.

In this study, patients with documented penicillin allergy had an increased incidence of broad spectrum antibiotic use, including the extended Gram positive spectrum antibiotics vancomycin and linezolid, which should be reserved for patients with suspected or known MRSA (or vancomycin resistant enterococci for linezolid).3839 Use of the most narrow spectrum antibiotic that is effective for a given infection is a cornerstone of evidence based treatment for infection and is responsible antibiotic stewardship.38 Antibiotic stewardship committees enforce this aim in the hospital setting, with evaluations for penicillin allergy occasionally included in stewardship efforts.40 This analysis emphasises the importance of performing outpatient antibiotic stewardship and the role that penicillin allergy evaluations might play. Although diagnostic testing for penicillin allergy was developed in the 1960s, and has recently garnered the support of a variety of professional organizations,384142 less than 0.1% of patients with a penicillin allergy label undergo confirmatory testing.15 Evaluation of penicillin allergy often involves a skin test, and if the result of skin testing is negative, a challenge dose of penicillin or amoxicillin is administered under medical observation.15 With these evaluation tools, evaluation of penicillin allergy has a more than 99% negative predictive value, takes less than three hours to perform, and costs about $220 (£165; €188; 2016 currency conversion).1543 Previous observational cohorts have shown that more than 90% of patients with listed penicillin allergies can be safely treated with penicillins.141540

Strengths and limitations of this study

In this study we used a representative population based cohort to increase the generalizability of our findings. Clinical data to characterise drug use, outcomes, and covariates were entered by physicians and captured electronically. The dataset used included granular allergy data linked to penicillin prescriptions and defined by type, severity, and certainty. Our study design used a comparator group who had recently been prescribed a penicillin but did not have a resultant penicillin allergy. Patients had high antibiotic use in the previous year since almost the entire cohort had recently had a penicillin (for infection) at baseline for cohort eligibility. Our GP practice based dataset could have missed the detection of some inpatient cases of MRSA and C difficile; however, these potential non-differential misclassifications would have biased our results towards the null, rendering our findings conservative. MRSA and C difficile were identified by physician diagnosis records. This approach has been successfully used in many previous epidemiologic studies,131920212223244445 as microbial infections such as MRSA and C difficile are made objectively using highly accurate microbiologic and serologic tests. Although we used composite outcomes for MRSA and C difficile that were not restricted to infections, it is unlikely that GPs would screen asymptomatic patients and more likely that diagnoses occurred in relevant clinical contexts where infections were suspected. Further, our findings remained consistent and strong when we restricted the analyses to code subgroups suggestive of infections. Additionally, MRSA carriage alone is an important outcome that confers an increased risk of MRSA infection,46 and indicates antibiotic resistance—a healthcare priority throughout the world.3947 Finally, by choosing to study only the first documentation of MRSA and C difficile, we ensured capture of only new colonization or infection, which are clinically important outcomes. Although we controlled for many known potential confounders in these data, our observational study cannot rule out potential unknown or residual confounding.

Conclusions

In this population based cohort study, a listed penicillin allergy was associated with a statistically significantly increased risk of MRSA and C difficile compared with patients matched by age, sex, and study entry time. Approximately one third to more than one half of this risk was attributed to use of non-β lactam antibiotics administered to outpatients. As infections with resistant organisms increase, systematic efforts to confirm or rule out the presence of true penicillin allergy may be an important public health strategy to reduce the incidence of MRSA and C difficile.

What is already known on this topic

Penicillin allergy is the most commonly documented drug allergy, reported by about 10% of patients

Although documented allergies impact prescribing behavior, a documented penicillin allergy does not often represent true, immediate hypersensitivity to penicillin

Previous studies have identified specific antibiotic uses that increase the risk of MRSA and Clostridium difficile

for C. difficile

Abstract

Conventional antibiotics for C. difficile infection (CDI) have mechanisms of action without organismal specificity, potentially perpetuating the dysbiosis contributing to CDI, making antisense approaches an attractive alternative. Here, three (APDE-8, CODE-9, and CYDE-21) novel cationic amphiphilic bolaamphiphiles (CABs) were synthesized and tested for their ability to form nano-sized vesicles or vesicle-like aggregates (CABVs), which were characterized based on their physiochemical properties, their antibacterial activities, and their toxicity toward colonocyte (Caco-2) cell cultures. The antibacterial activity of empty CABVs was tested against cultures of E. coli, B. fragilis, and E. faecalis, and against C. difficile by “loading” CABVs with 25-mer antisense oligonucleotides (ASO) targeting dnaE. Our results demonstrate that empty CABVs have minimal colonocyte toxicity until concentrations of 71 µM, with CODE-9 demonstrating the least toxicity. Empty CABVs had little effect on C. difficile growth in culture (MIC90 ≥ 160 µM). While APDE-8 and CODE-9 nanocomplexes demonstrated high MIC90 against C. difficile cultures (>300 µM), CYDE-21 nanocomplexes demonstrated MIC90 at CABV concentrations of 19 µM. Empty CABVs formed from APDE-8 and CODE-9 had virtually no effect on E. coli, B. fragilis, and E. faecalis across all tested concentrations, while empty CYDE-21 demonstrated MIC90 of >160 µM against E. coli and >40 µM against B. fragilisand E. faecalis. Empty CABVs have limited antibacterial activity and they can deliver an amount of ASO effective against C. difficile at CABV concentrations associated with limited colonocyte toxicity, while sparing other bacteria. With further refinement, antisense therapies for CDI may become a viable alternative to conventional antibiotic treatment.

Introduction

C. difficile infection (CDI) is the most frequently reported nosocomial bacterial infection [1] in the United States, accounting for more than 450,000 new cases annually and for more than four billion dollars in CDI-attributable annual health care costs [2]. CDI has a strong reliance on intestinal dysbiotic states, which, when combined with the presence of C. difficile in the human gut, represents the most common pathogenesis for CDI. The high prevalence of this infection is, in large part, due to formidable recurrence rates of 15–25% following first treatment [3] with conventional antibiotics (CAs). CAs have long been recognized as the most important risk factor for the development of CDI [4], due to their mechanisms of action lacking organismal specificity, leading to widespread changes in gut ecology [5], which can lead to CDI by disrupting the gut microbial community. Given the important role of intestinal dysbiosis in the development of CDI, there has also been recent interest in studying the effects of difficile-directed conventional antibiotics on the bacterial and fungal communities of human subjects being treated for CDI, as a way of potentially explaining the high persistence and recurrence rates of this disease. These more recent data [6] suggest that even difficile-directed conventional antibiotics could potentially contribute to the perpetuation of dysbiotic states, which in turn could perpetuate CDI, potentially leading to even primary treatment failures.

There has been previous [7, 8] interest in the development of antisense therapies to treat bacterial infections, in part due to concerns regarding antibiotic resistance to traditional drugs. Given the dependence of CDI on dysbiotic states, approaches using therapeutic antisense oligonucleotides (ASO) complimentary to specific C. difficile mRNAs could limit or prevent the expression of important bacterial genes leading to bacterial death, all while sparing other organisms. This approach would offer significant advantages over CAs, especially in terms of a more limited impact on gut microbial communities. Developing clinically effective antisense therapies targeting a Gram-positive organism requires several elements. Since antisense oligonucleotides will not be efficiently introduced into bacteria without assistance given the presence of both a cell membrane and a thick cell wall, a carrier molecule must be used to deliver the ASO. This carrier must complex with the ASO strongly enough to concentrate it, to protect it from degradation in the extracellular environment, and to focus its delivery on its target cell. In order to accomplish these activities, the carrier-ASO complex itself must be stable in the in vivo environment of the gut. Once at the cell, the carrier must be able to release its cargo. Simultaneously, the carrier must demonstrate both limited gut toxicity and limited antibacterial activity at the doses required to effectively treat the target bacteria.

Our group published the first [9] in vitro data for antisense therapies against CDI by complexing cyclohexyl dequalinium analogs to various ASO-targeting essential C. difficile genes. However, since dequalinium has both antibacterial activity as well as toxicity at higher doses, a better delivery compound for ASO is required if antisense approaches to CDI are to be further developed. Here, we report our data on vesicles formed from novel cationic amphiphilic bolaamphiphiles (CABs) as carriers for chimeric 25-mer 2′-O-methyl phosphorothioate ASO. CABs, characteristic of all bola-like compounds, have hydrophilic, positively charged end groups separated by a hydrophobic linker chain. This molecular structure enables CABs to form nano-sized vesicle-like aggregates (CABVs), which in turn allow them to complex with negatively charged oligonucleotides in addition to promoting electrostatic interactions with bacterial cell membranes for intracellular delivery of ASO. The synthesis, physiochemical properties, toxicity, and antibacterial properties of three novel CABs and their respective CABVs are described, and their specificity for C. difficile compared to several other organisms is also provided.

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Clostridium difficile (C. diff) is among one of the top 18-drug-resistant threats to the United States according to the Centers for Disease Control and Prevention, responsible for around 250,000 infections on an annual basis and 14,000 deaths.

When it comes to diagnosis, microbiological testing of stool samples is often used. However, a new study suggests that for simple PCR-based detection of C. diff, dry rectal swabs were an effective substitute for the use of stool samples.

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“With this study, we proved that rectal swabs for the diagnosis of C. diff infection by PCR can replace the actually used stool samples,” study author Nathalie Jazmati, MD, University Hospital of Cologne, told our sister publication MD Magazine. “That will be more convenient for both patients and health care workers. Nevertheless, this was only a small study and our results have to be confirmed in a bigger clinical trial.”

In an effort to examine methods other than the analysis of stool specimens for C. diff confirmation, a research team from Germany examined the way rectal swabs with liquid transport medium and nylon flocked dry swabs performed for the detection of C. diff; they also evaluated the impact of storage temperature on the swabs.

For their study, the researchers collected 60 clinical stool samples that tested positive for C. diff by PCR and used them to simulate rectal swabs. Then, researchers dipped both wet and dry swabs into the stool and tested by PCR 3 times.

The first test took place immediately after the simulation “swab,” then, after 1 month and 3 months storage at -80°C. When the researchers tested the frozen samples, they first thawed them at room temperature for 15 minutes and the liquid swabs were vortexed for 30 seconds.

Testing all of the dry swabs 100% successfully detected C. diff, an equal rate of the stool sample testing; this proved true for all 3 phases of testing, and the researchers learned that no significant differences were found on the samples after they were frozen and thawed.

The detection rate for the other 30 liquid swabs was lower, at 83.2% accuracy. However, the researchers determined the temperature and the freezing and thawing of these samples did not have any significant impact.

The authors added that their results fall in line with other studies that tested PCR from rectal swabs in the detection of C. diff. The idea of using rectal swabs instead of stool samples isn’t new—it dates back to 1987.

Liquid swabs are currently cleared by the US Food and Drug Administration (FDA) for transport and the culture of gastrointestinal pathogens, the study authors continued, but it is not FDA approved for use with any molecular gastrointestinal assays.

In the future, dry swabs would “be appropriate and can probably speed up and facilitate the diagnosis of C. diff infection,” the researchers wrote, but warned, “nevertheless, using single step PCR-based detection of C. diff may lead to overdiagnosis of C. diff infection due to the high sensitivity but lower specificity of PCR.”

That marks a heightened importance for the careful clinical evaluation of the patient: Are they an asymptomatic carrier? Is there another reason for the patient’s diarrhea? Do they truly have a C. diff infection? All important questions to continue to ask.

While liquid swabs cannot substitute for the two-step laboratory diagnosis of C. diff, the researchers believe that their study shows the dry swab is a suitable alternative to stool sample testing.

Using those hot-air hand dryers in restrooms actually spread bacteria, including fecal bacteria on your hands, according to a new study conducted at UConn.

“In most institutions, toilets don’t have lids and when you flush them you get a little bit of an aerosol,” said Dr. Peter Setlow, a molecular biology and biophysics professor at UConn Health.

So, where does that toilet plume go?

“The hand dryers grab that air in the bathroom and accelerate it,” Setlow said.

During the study, plates were placed under hand dryers to collect the bacteria being blown out of the hand dryer.

“We’d get up to 150, 200 individual bacterial colonies and obviously different because they were different colors, different shapes, different smell,” Setlow said.

They tested 36 bathrooms multiple times and got similar results. The study was done in a research area in the academic building at UConn Health in Farmington not open to the public. However, Setlow said hand dryers in most public bathrooms work the same way.

“The bacteria that are blown from the hand dryers are coming from the bathroom air,” said Setlow.

Setlow has been studying molecular biology for 50 years. He said of the nearly 500 papers he’s helped author, this one, published in Applied and Environmental Microbiology, has gotten the most attention.

People who spoke with NBC Connecticut said news of the study may make them change their habits.

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